Concomitant production of nitric oxide and superoxide in human macrophages

Many harmful effects of nitric oxide are caused by the reaction of NO with superoxide anion. The present study was carried out to find out the concomitant production of superoxide and to investigate a suitable inhibitor of NO, which is produced by iNOS. THP-1 cells were differentiated into macrophages by PMA and cytokine. Addition of L-NAME showed decrement in superoxide production. Addition of apocynin, aminoguanidine or ONO 1714 brought about a significant reduction in superoxide production. The expressions of p67 and p47(phox) were reduced by the addition of apocynin, aminoguanidine or ONO 1714 whereas xanthine oxidase and cyclooxygenase did not have a major role in superoxide production. The results of the present study show that iNOS and NADPH oxidase play an important role in superoxide release. It suggests that addition of iNOS inhibitor together with apocynin may be more effective in case of therapeutic application in disease conditions like atherosclerosis.     

Homocysteine stimulates phosphorylation of NADPH oxidase p47phox and p67phox subunits in monocytes via protein kinase Cbeta activation

Hyperhomocysteinaemia is an independent risk factor for cardiovascular diseases due to atherosclerosis. The development of atherosclerosis involves reactive oxygen species-induced oxidative stress in vascular cells. Our previous study [Wang and O (2001) Biochem. J. 357, 233-240] demonstrated that Hcy (homocysteine) treatment caused a significant elevation of intracellular superoxide anion, leading to increased expression of chemokine receptor in monocytes. NADPH oxidase is primarily responsible for superoxide anion production in monocytes. In the present study, we investigated the molecular mechanism of Hcy-induced superoxide anion production in monocytes. Hcy treatment (20-100 microM) caused an activation of NADPH oxidase and an increase in the superoxide anion level in monocytes (THP-1, a human monocytic cell line). Transfection of cells with p47phox siRNA (small interfering RNA) abolished Hcy-induced superoxide anion production, indicating the involvement of NADPH oxidase. Hcy treatment resulted in phosphorylation and subsequently membrane translocation of p47phox and p67phox subunits leading to NADPH oxidase activation. Pretreatment of cells with PKC (protein kinase C) inhibitors Ro-32-0432 (bisindolylmaleimide XI hydrochloride) (selective for PKCalpha, PKCbeta and PKCgamma) abolished Hcy-induced phosphorylation of p47phox and p67phox subunits in monocytes. Transfection of cells with antisense PKCbeta oligonucleotide, but not antisense PKCalpha oligonucleotide, completely blocked Hcy-induced phosphorylation of p47phox and p67phox subunits as well as superoxide anion production. Pretreatment of cells with LY333531, a PKCbeta inhibitor, abolished Hcy-induced superoxide anion production. Taken together, these results indicate that Hcy-stimulated superoxide anion production in monocytes is regulated through PKC-dependent phosphorylation of p47phox and p67phox subunits of NADPH oxidase. Increased superoxide anion production via NADPH oxidase may play an important role in Hcy-induced inflammatory response during atherogenesis.     

NADPH oxidase contributes to renal damage and dysfunction in Dahl salt-sensitive hypertension

The goal of this study was to test the hypothesis that NADPH oxidase contributes importantly to renal cortical oxidative stress and inflammation, as well as renal damage and dysfunction, and increases in arterial pressure. Fifty-four 7- to 8-wk-old Dahl salt-sensitive (S) or R/Rapp strain rats were maintained for 5 wk on a high sodium (8%) or high sodium + apocynin (1.5 mmol/l in drinking water). Arterial and venous catheters were implanted on day 21. By day 35 in the high-Na S rats, mRNA expression of renal cortical gp91phox, p22phox, p47phox, and p67phox NADPH subunits in S rats increased markedly, and treatment of high-Na S rats with the NADPH oxidase inhibitor apocynin resulted in significant decreases in mRNA expression of these NADPH oxidase subunits. At the same time, in apocynin-treated S rats 1) renal cortical GSH/GSSG ratio increased, 2) renal cortical O2(.-) release and NADPH oxidase activity decreased, and 3) renal glomerular and interstitial damage markedly fell. Apocynin also decreased renal cortical monocyte/macrophage infiltration, and apocynin, but not the xanthine oxidase inhibitor allopurinol, attenuated decreases in renal hemodynamics and lowered arterial pressure. These data suggest that NADPH oxidase plays an important role in causing renal cortical oxidative stress and inflammation, which lead to decreases in renal hemodynamics, renal cortical damage, and increases in arterial pressure.     

Activation of NADPH oxidase and extracellular superoxide production in seizure-induced hippocampal damage

We sought to determine whether the extracellular compartment contributed to seizure-induced superoxide (O2*-) production and to determine the role of the NADPH oxidase complex as a source of this O2*- production. The translocation of NADPH oxidase subunits (p47phox, p67phox and rac1) was assessed by immunoblot analysis and NADPH-driven O2*- production was measured using 2-(4-hydroxybenzyl)-6-(4-hydroxyphenyl)-8-benzyl-3,7-dihydroimidazo [1,2-alpha] pyrazin-3-one-enhanced chemiluminescence. Kainate-induced status epilepticus resulted in a time-dependent translocation of NADPH oxidase subunits (p47phox, p67phox and rac-1) from hippocampal cytosol to membrane fractions. Hippocampal membrane fractions from kainate-injected rats showed increased NADPH-driven and diphenylene iodonium-sensitive O2*- production in comparison to vehicle-treated rats. The time-course of kainate-induced NADPH oxidase activation coincided with microglial activation in the rat hippocampus. Finally, kainate-induced neuronal damage and membrane oxygen consumption were inhibited in mice overexpressing extracellular superoxide dismutase. These results suggest that seizure activity activates the membrane NADPH oxidase complex resulting in increased formation of O2*-.     

Rotenone activates phagocyte NADPH oxidase by binding to its membrane subunit gp91phox

Rotenone, a widely used pesticide, reproduces parkinsonism in rodents and associates with increased risk for Parkinson disease. We previously reported that rotenone increased superoxide production by stimulating the microglial phagocyte NADPH oxidase (PHOX). This study identified a novel mechanism by which rotenone activates PHOX. Ligand-binding assay revealed that rotenone directly bound to membrane gp91(phox), the catalytic subunit of PHOX; such binding was inhibited by diphenyleneiodonium, a PHOX inhibitor with a binding site on gp91(phox). Functional studies showed that both membrane and cytosolic subunits were required for rotenone-induced superoxide production in cell-free systems, intact phagocytes, and COS7 cells transfected with membrane subunits (gp91(phox)/p22(phox)) and cytosolic subunits (p67(phox) and p47(phox)). Rotenone-elicited extracellular superoxide release in p47(phox)-deficient macrophages suggested that rotenone enabled activation of PHOX through a p47(phox)-independent mechanism. Increased membrane translocation of p67(phox), elevated binding of p67(phox) to rotenone-treated membrane fractions, and coimmunoprecipitation of p67(phox) and gp91(phox) in rotenone-treated wild-type and p47(phox)-deficient macrophages indicated that p67(phox) played a critical role in rotenone-induced PHOX activation via its direct interaction with gp91(phox). Rac1, a Rho-like small GTPase, enhanced p67(phox)-gp91(phox) interaction; Rac1 inhibition decreased rotenone-elicited superoxide release. In conclusion, rotenone directly interacted with gp91(phox); such an interaction triggered membrane translocation of p67(phox), leading to PHOX activation and superoxide production.     

NADPH oxidase and apoptosis in cerulein-stimulated pancreatic acinar AR42J cells

Apoptosis linked to oxidative stress has been implicated in pancreatitis. We investigated whether NADPH oxidase mediates apoptosis in cerulein-stimulated pancreatic acinar AR42J cells. We report here that cerulein treatment resulted in the activation of NADPH oxidase, as determined by ROS production, translocation of cytosolic subunits p 47(phox) and p 67(phox) to the membrane, and interaction between NADPH oxidase subunits. Cerulein induced Ca(2+) oscillation, the expression of apoptotic genes p53 and bax, and apoptotic indices (DNA fragmentation, TUNEL staining, caspase 3 activity, decrease in cell viability) in AR42J cells. Treatment with a Ca(2+) chelator, BAPTA-AM, or transfection with antisense oligonucleotides for NADPH oxidase subunits p22(phox) and p 47(phox) inhibited cerulein-induced ROS production, translocation of NADPH oxidase cytosolic subunits p 47(phox) and p 67(phox) to the membrane, and the expression of apoptotic genes and apoptotic indices, as compared to the cells without treatment and those transfected with the corresponding sense oligonucleotides. These results indicate that NADPH oxidase may mediate ROS-induced apoptosis in pancreatic acinar cells in a Ca(2+)-dependent manner.     

17beta-estradiol inhibits NADPH oxidase activity through the regulation of p47phox mRNA and protein expression in THP-1 cells

In this report, we demonstrate that NADPH oxidase is activated by tumor necrosis factor-alpha (TNF-alpha) plus interferon-gamma (IFN-gamma) in human monocytic cells (THP-1 cells) differentiated with phorbol ester (PMA) and that physiological concentration of 17beta-estradiol inhibits NADPH oxidase activity in THP-1 cells stimulated with TNF-alpha plus IFN-gamma. This effect is mediated by estrogen receptor based on estrogen receptor antagonist (ICI 182, 780) that diminishes inhibition by 17beta-estradiol. This inhibition is specific in 17beta-estradiol because 17alpha-estradiol, testosterone and progesterone do not inhibit NADPH oxidase activity. Activation of NADPH oxidase induced by TNF-alpha plus IFN-gamma is caused by up-regulation of p47(phox) (cytosolic component of NADPH oxidase) expression. 17beta-Estradiol prevents the up-regulation of p47(phox) mRNA and protein expression. This prevention of p47(phox) expression depends on the inhibition of NF-kappaB activation. Our results implicate that 17beta-estradiol has an anti-atherosclerotic effects through the improvement of nitric oxide (NO) bioavailability caused by the regulation of superoxide (O(2)(-)) production.     

Ceramide-induced activation of NADPH oxidase and endothelial dysfunction in small coronary arteries

We tested the hypothesis that ceramide induces endothelial dysfunction in small coronary arteries via NADPH oxidase-mediated superoxide and resulting peroxynitrite formation. With the use of dihydroethidium as a superoxide indicator, C(2)-ceramide was found to increase superoxide production in the endothelial cells of small coronary arteries, which was inhibited by the NADPH oxidase inhibitors N-vanillylnonanamide, apocynin, and diphenylene iodonium. NADPH oxidase expression was confirmed in endothelial cells, as indicated by the immunoblotting of its subunits gp91(phox) and p47(phox). C(2)-ceramide increased NADPH oxidase activity by 52%, which was blocked by NADPH oxidase inhibitors but not by inhibitors of NO synthase, xanthine oxidase, and mitochondrial electron transport chain enzymes. By Western blot analysis, ceramide-induced NADPH oxidase activation was found to be associated with the translocation of p47(phox) to the membrane. In isolated and pressurized small coronary arteries, N-vanillylnonanamide, apocynin, or uric acid, a peroxynitrite scavenger, largely restored the inhibitory effects of ceramide on bradykinin- and A-23187-induced vasorelaxation. With the use of nitrotyrosine as a marker, C(2)-ceramide was found to increase peroxynitrite in small coronary arteries, which could be blocked by uric acid. We conclude that NADPH oxidase-mediated superoxide production and subsequent peroxynitrite formation mediate ceramide-induced endothelial dysfunction in small coronary arteries.     

Tumor necrosis factor-alpha increases airway smooth muscle oxidants production through a NADPH oxidase-like system to enhance myosin light chain phosphorylation and contractility

Tumor necrosis factor plays a critical role in airway smooth muscle hyperresponsiveness observed in asthma. However, the mechanisms underlying this phenomenon are poorly understood. We investigated if tumor necrosis factor-stimulated airway smooth muscle produced reactive oxygen species, leading to muscular hyperresponsiveness. Tumor necrosis factor increased intracellular and extracellular oxidants production in guinea pig airway smooth muscle cells and tissue homogenates. This production was abolished by inhibitors of NADPH oxidase (diphenylene iodinium or apocynin) and was enhanced by NADPH, whereas inhibitors of mitochondrial respiratory chain, nitric-oxide synthase, cyclooxygenase, and xanthine oxidase had no effect. NADPH oxidase subunits p22(phox) and p47(phox) were detected in smooth muscle cells and tissue homogenates by Western blot, immunohistochemistry, and spectral analysis. Furthermore, oxidants production was significantly reduced by transient transfection of smooth muscle cells with p22(phox) antisense oligonucleotides. Intracellular antioxidants and diphenylene iodinium abolished tumor necrosis factor-induced muscular hyperresponsiveness and increased in phosphorylation of the myosin light chain. Finally, NADPH oxidase subunits p22(phox) and p47(phox) were also detected in human airway smooth muscle. Collectively, these results demonstrate that tumor necrosis factor-stimulated airway smooth muscle produces oxidants through a NADPH oxidase-like system, which plays a pivotal role in muscle hyperresponsiveness and myosin light chain phosphorylation.     

Impact of androgen-induced oxidative stress on hypertension in male SHR

Men have higher blood pressure than women, and androgens and oxidative stress have been implicated as playing roles in this sexual dimorphism. The spontaneously hypertensive rat (SHR) is an animal model of both androgen- and oxidative stress-mediated hypertension. Therefore, the present studies were performed to test the hypothesis that androgens cause hypertension in SHR in part by stimulating superoxide production via NADPH oxidase. Castration of male SHR reduced blood pressure by 15% and attenuated both basal and NADPH-stimulated superoxide production in kidney cortical homogenates. Expression of p47(phox) and gp91(phox) but not p22(phox) subunits of NADPH oxidase were significantly lower in kidney cortex from castrated males compared with intact males. Moreover, inhibition of NADPH oxidase with apocynin caused approximately 15 mmHg reduction in blood pressure and reduced basal and NADPH-stimulated superoxide production in intact male SHR, but had no effect on blood pressure or superoxide production in castrated males. These data support the hypothesis that androgens cause oxidative stress and thereby increase blood pressure in male SHR via an NADPH oxidase-dependent mechanism.     

Inorganic arsenic activates reduced NADPH oxidase in human primary macrophages through a Rho kinase/p38 kinase pathway

Inorganic arsenic is an immunotoxic environmental contaminant to which millions of humans are chronically exposed. We recently demonstrated that human primary macrophages constituted a critical target for arsenic trioxide (As(2)O(3)), an inorganic trivalent form. To specify the effects of arsenic on macrophage phenotype, we investigated in the present study whether As(2)O(3) could regulate the activity of NADPH oxidase, a major superoxide-generating enzymatic system in human phagocytes. Our results show that superoxide levels were significantly increased in a time-dependent manner in blood monocyte-derived macrophages treated with 1 muM As(2)O(3) for 72 h. Concomitantly, As(2)O(3) induced phosphorylation and membrane translocation of the NADPH oxidase subunit p47(phox) and it also increased translocation of Rac1 and p67(phox). Apocynin, a selective inhibitor of NADPH oxidases, prevented both p47(phox) translocation and superoxide production. NADPH oxidase activation was preceded by phosphorylation of p38-kinase in As(2)O(3)-treated macrophages. The p38-kinase inhibitor SB-203580 prevented phosphorylation and translocation of p47(phox) and subsequent superoxide production. Pretreatment of macrophages with the Rho-kinase inhibitor Y-27632 was found to mimic inhibitory effects of SB-203580 and to prevent As(2)O(3)-induced phosphorylation of p38 kinase. Treatment with As(2)O(3) also resulted in an increased secretion of the proinflammatory chemokine CCL18 that was fully inhibited by both apocynin and SB-203580. Taken together, our results demonstrate that As(2)O(3) induced a marked activation of NADPH oxidase in human macrophages, likely through stimulation of a Rho-kinase/p38-kinase pathway, and which may contribute to some of the deleterious effects of inorganic arsenic on macrophage phenotype.     

Superoxide anion is elevated in sympathetic neurons in DOCA-salt hypertension via activation of NADPH oxidase

Superoxide anion (O2-*) production is elevated in sympathetic ganglion neurons and in the vasculature of hypertensive animals; however, it is not known what enzymatic pathway(s) are responsible for O2-* production. To determine the pathway(s) of O2-* production in sympathetic neurons, we examined the presence of mRNA of NADPH oxidase subunits in sympathetic ganglionic neurons and differentiated PC-12 cells. The mRNAs for NADPH oxidase subunits p47phox, p22phox, gp91phox, and NOX1 were present in sympathetic neurons and PC-12 cells, whereas the NOX4 homologue was present in sympathetic neurons but not PC-12 cells. Freshly dissociated celiac ganglion neurons from normal rats and PC-12 cells produced O2-* when treated with the PKC activator PMA; O2-* production increased by 317% and 254%, respectively. The PMA-evoked increases were reduced by pretreatment with the NADPH oxidase inhibitor apocynin. These findings indicate that NADPH oxidase is the primary source of O2-* in sympathetic ganglion neurons. When celiac ganglia from hypertensive rats were incubated with apocynin, O2-* levels were reduced to the same levels as normotensive animals, indicating that NADPH oxidase activity accounted for the elevated O2-* levels in hypertensive animals. To test this latter finding, we compared NADPH oxidase activity in extracts of prevertebral sympathetic ganglia of DOCA-salt hypertensive rats and sham-operated rats. NADPH oxidase activities were 49.9% and 78.6% higher in sympathetic ganglia of DOCA rats compared with normotensive controls when using beta-NADH and beta-NADPH as substrates, respectively. Thus elevated O2-* levels in hypertension may be a result of the increased activity of NADPH oxidase in postganglionic sympathetic neurons.     

Angiotensin II-induced NADPH oxidase activation impairs insulin signaling in skeletal muscle cells

The renin-angiotensin system (RAS) and reactive oxygen species (ROS) have been implicated in the development of insulin resistance and its related complications. There is also evidence that angiotensin II (Ang II)-induced generation of ROS contributes to the development of insulin resistance in skeletal muscle, although the precise mechanisms remain unknown. In the present study, we found that Ang II markedly enhanced NADPH oxidase activity and consequent ROS generation in L6 myotubes. These effects were blocked by the angiotensin II type 1 receptor blocker losartan, and by the NADPH oxidase inhibitor apocynin. Ang II also promoted the translocation of NADPH oxidase cytosolic subunits p47phox and p67phox to the plasma membrane within 15 min. Furthermore, Ang II abolished insulin-induced tyrosine phosphorylation of insulin receptor substrate 1 (IRS1), activation of protein kinase B (Akt), and glucose transporter-4 (GLUT4) translocation to the plasma membrane, which was reversed by pretreating myotubes with losartan or apocynin. Finally, small interfering RNA (siRNA)-specific gene silencing targeted specifically against p47phox (p47siRNA), in both L6 and primary myotubes, reduced the cognate protein expression, decreased NADPH oxidase activity, restored Ang II-impaired IRS1 and Akt activation as well as GLUT4 translocation by insulin. These results suggest a pivotal role for NADPH oxidase activation and ROS generation in Ang II-induced inhibition of insulin signaling in skeletal muscle cells.     

Mapping of functional domains in the p22(phox) subunit of flavocytochrome b(559) participating in the assembly of the NADPH oxidase complex by "peptide walking".

The superoxide-generating NADPH oxidase complex of phagocytes consists of a membranal heterodimeric flavocytochrome (cytochrome b(559)), composed of gp91(phox) and p22(phox) subunits, and four cytosolic proteins, p47(phox), p67(phox), p40(phox), and the small GTPase Rac (1 or 2). All redox stations involved in electron transport from NADPH to oxygen are located in gp91(phox). NADPH oxidase activation is the consequence of assembly of cytochrome b(559) with cytosolic proteins, a process reproducible in a cell-free system, consisting of phagocyte membranes, and recombinant cytosolic components, activated by an anionic amphiphile. p22(phox) is believed to act as a linker between the cytosolic components and gp91(phox). We applied "peptide walking" to mapping of domains in p22(phox) participating in NADPH oxidase assembly. Ninety one synthetic overlapping pentadecapeptides, spanning the p22(phox) sequence, were tested for the ability to inhibit NADPH oxidase activation in the cell-free system and to bind individual cytosolic NADPH oxidase components. We conclude the following. 1) The p22(phox) subunit of cytochrome b(559) serves as an anchor for both p47(phox) and p67(phox). 2) p47(phox) binds not only to the proline-rich region, located at residues 151-160 in the cytosolic C terminus of p22(phox), but also to a domain (residues 51-63) located on a loop exposed to the cytosol. 3) p67(phox) shares with p47(phox) the ability to bind to the proline-rich region (residues 151-160) and also binds to two additional domains, in the cytosolic loop (residues 81-91) and at the start of the cytosolic tail (residues 111-115). 4) The binding affinity of p67(phox) for p22(phox) peptides is lower than that of p47(phox). 5) Binding of both p47(phox) and p67(phox) to proline-rich p22(phox) peptides occurs in the absence of an anionic amphiphile. A revised membrane topology model of p22(phox) is proposed, the core of which is the presence of a functionally important cytosolic loop (residues 51-91).     

D5 dopamine receptor regulation of reactive oxygen species production, NADPH oxidase, and blood pressure

Activation of D1-like receptors (D1 and/or D5) induces antioxidant responses; however, the mechanism(s) involved in their antioxidant actions are not known. We hypothesized that stimulation of the D5 receptor inhibits NADPH oxidase activity, and thus the production of reactive oxygen species (ROS). We investigated this issue in D5 receptor-deficient (D5-/-) and wild-type (D5+/+) mice. NADPH oxidase protein expression (gp91(phox), p47(phox), and Nox 4) and activity in kidney and brain, as well as plasma thiobarbituric acid-reactive substances (TBARS) were higher in D5-/- than in D5+/+ mice. Furthermore, apocynin, an NADPH oxidase inhibitor, normalized blood pressure, renal NADPH oxidase activity, and plasma TBARS in D5-/- mice. In HEK-293 cells that heterologously expressed human D5 receptor, its agonist fenoldopam decreased NADPH oxidase activity, expression of one of its subunits (gp91(phox)), and ROS production. The inhibitory effect of the D5 receptor activation on NADPH oxidase activity was independent of cAMP/PKA but was partially dependent on phospholipase D2. The ability of D5 receptor stimulation to decrease ROS production may explain, in part, the antihypertensive action of D5 receptor activation.     

Adventitial delivery of dominant-negative p67phox attenuates neointimal hyperplasia of the rat carotid artery

Several essential components of NADPH oxidase, including p22phox, gp91phox (nox2) and its homologs nox1 and nox4, p47phox, p67phox, and rac1, are present in the vasculature. We previously reported that p67phox is essential for adventitial fibroblast NADPH oxidase O2- production. Thus we postulated that inhibition of adventitial p67phox activity would attenuate angioplasty-induced hyperplasia. To test this hypothesis, we treated the adventitia of carotid arteries with a control adenovirus (Ad-control), a virus expressing dominant-negative p67phox (Ad-p67dn), or a virus expressing a competitive peptide (gp91ds) targeting the p47phox-gp91phox interaction (Ad-gp91ds). Common carotid arteries (CCAs) from male Sprague-Dawley rats were transfected with Ad-control, Ad-p67dn, or Ad-gp91ds in pluronic gel. After 2 days, a 2-F (Fogarty) catheter was used to injure CCAs in vivo. After 14 days, CCAs were perfusion-fixed and analyzed. In 13 experiments, digital morphometry suggested a reduction of neointimal hyperplasia with Ad-p67dn compared with Ad-control; however, the reduction did not reach statistical significance (P = 0.058). In contrast, a significant reduction was achieved with Ad-gp91ds (P = 0.006). No changes in medial area or remodeling were observed with either treatment. Moreover, adventitial fibroblast proliferation in vitro was inhibited by Ad-gp91ds but not by Ad-p67dn, despite confirmation that Ad-p67dn inhibits NADPH oxidase in fibroblasts. These data appear to suggest that a multicomponent vascular NADPH oxidase plays a role in neointimal hyperplasia. However, inhibition of p47phox may be more effective than inhibition of p67phox at attenuating neointimal growth.     

Intracellular localization and preassembly of the NADPH oxidase complex in cultured endothelial cells

The phagocyte-type NADPH oxidase expressed in endothelial cells differs from the neutrophil enzyme in that it exhibits low level activity even in the absence of agonist stimulation, and it generates intracellular reactive oxygen species. The mechanisms underlying these differences are unknown. We studied the subcellular location of (a) oxidase subunits and (b) functionally active enzyme in unstimulated endothelial cells. Confocal microscopy revealed co-localization of the major oxidase subunits, i.e. gp91(phox), p22(phox), p47(phox), and p67(phox), in a mainly perinuclear distribution. Plasma membrane biotinylation experiments confirmed the predominantly (>90%) intracellular distribution of gp91(phox) and p22(phox). After subcellular protein fractionation, approximately 50% of the gp91(phox) (91-kDa band), p22(phox), p67(phox), and p40(phox) pools and approximately 30% of the p47(phox) were present in the 1475 x g ("nucleus-rich") fraction. Likewise, approximately 50% of total NADPH-dependent O(2)() production (assessed by lucigenin (5 microm) chemiluminescence) was found in the 1475 x g fraction. Co-immunoprecipitation studies and measurement of NADPH-dependent reactive oxygen species production (cytochrome c reduction assay) demonstrated that p22(phox), gp91(phox), p47(phox), p67(phox), and p40(phox) existed as a functional complex in the cytoskeletal fraction. These results indicate that, in contrast to the neutrophil enzyme, a substantial proportion of the NADPH oxidase in unstimulated endothelial cells exists as a preassembled intracellular complex associated with the cytoskeleton.